The invention relates to a peptide vector which can achieve gene transfer without cell specificity and does not induce host immune responses.
It is known that viral vectors have been mainly used for introducing genes into a living body, by using their cell endosmosis ability. These viral vectors include, for example, adenovirus, herpes virus, retrovirus, and the like.
Adenovirus is a non-enveloped double-stranded DNA virus, and causes insignificant upper respiratory tract infections, keratoconjunctivitis, enterogastritis, and the like in human. The adenovirus genomes are approximately 36 kb, and easy to handle by using conventional recombinant DNA technology. Cell endosmosis of virus is initialized by binding of a fiber knob protein of adenovirus to Coxsackie & Adenovirus receptor (CAR) on the cell surface. Subsequently, via the interactions between integrins (α vβ 3, α vβ 5) on the cell surface and the capsid penton base, the virion is introduced into the cell by the clathrin coated endocytosis. Then, the conforrnational changes of virion capsid proteins are derived, as the low pH condition of the endosome, allowing the release of the virion capsid proteins to the cytoplasm. Following the protein release, the capsid proteins translocate to the nucleus, where its replication and transcription are carried out. Transcription of genes can be divided into two different types, for early genes (E) which are expressed before the replication, and late genes (L) which are expressed after the replication. Adenovirus used for gene therapy lacks the E1 gene area, resulting incomplete replication, thereby being used in a form containing other DNAs inserted thereto. Therefore, the recombinant adenovirus vectors can be cultured to increase, in cell lines such as HEK293 which can express E1 genes continuously.
Retrovirus with envelope is a single stranded RNA virus having a diploid genome of about 7˜10 kb, and comprises the four gene groups called gag, pro, pol, and env, respectively. Each of the gene group encodes the structural capsid protein, viral protease, integrase, reverse transcriptase, envelope and glycoprotein, etc. The retrovirus has a packing signal (ψ) referred as long-terminal repeat (LTR), and a cis-acting sequence. Retrovirus infection of a cell can be achieved by primarily, binding of the envelope glycoprotein to its cell surface receptor, and subsequent fusing of the virus envelope with the cell membrane, thereby internalizing the capsid nucleus into the cell. Once the capsid has entered into the cytoplasm, the reverse transcriptase inside the capsid produces double stranded proviral genome, which forms a complex with an integrase and moves to the nuclear membrane. When the nuclear membrane disappears during mitosis, the complex enters into the nucleus. The proviral genome introduced into the nucleus inserts into chromosome of the host by means of an integrase, to express the viral genome by using the transcription apparatus of the host. The recombinant retrovirus vector does not express any viral gene, that makes this distinguished from the above-mentioned adenovirus vectors, as all of its genes are replaced to marker or therapeutic genes except LTRs and ψ sequence. To cultivate such recombinant retrovirus, the viral genes of gag, pol, and env should be expressed in a trans form, which can be achieved by using cell lines that express these genes in stable manner.
Adeno-binding virus belongs to a parvoviridae family, and is a simple virus having short single stranded DNA genome. The adeno-binding virus is comprised of two open reading frames (ORF) which are rep (control) and cap (structure), and two inverted terminal repeats (ITRs). The inverted terminal repeats are the only part needed for encapsidation and integration into the host genome, being stably integrated into the 19th human chromosome. However, the virus does not have self-growth ability, so it can be only grown under the presence of helper virus such as adenovirus or herpesvirus, etc. The recombinant adeno-binding virus is formed by the co-transfection with a plasmid having a transcription unit inserted between the inverted terminal repeats, and a plasmid containing a rep and cap open reading frames, and in this time, a sub-infection with a helper virus such as adenovirus is needed. After purifying process of recombinant adeno-binding virus, it can be used for gene therapy applications.
Herpes simplex virus-1 is a double stranded DNA virus having envelope. It encodes 80 or more genes from its 152 kb genomes, and has a significantly wide range of hosts as its envelope glycoproteins (gB, gC) bind to the extracellular heparan sulphate, that is discovered from the all kinds of cell membrane. When the virus is being entered into the host cell, the virus envelope glycoprotein gD and the fibroblast growth factor (FGF) receptor of the host are necessary. Herpes simplex virus vector can be divided into two types, a recombinant herpes simplex virus vector and an amplicon vector, wherein the recombinant herpes simplex virus vector has a transcription unit directly inserted in its genome, and the amplicon vector is a plasmid infected with a helper virus, wherein the said plasmid contains the transcription unit, replication origin, and packing signal. The amplicon vector is subjected to the rolling circle replication, producing a herpes simplex virus having an insertion of multiple copy genes during packing procedure.
Those above-mentioned virus vectors have their own advantages and disadvantages. Common problems to these above virus vectors include the limitation of the cell ranges, which could be infected by them, i.e. the limitation in cells which could receive them, and the inactivation by immune responses of host. As these types of vectors have such limitations, they are not applicable to all kinds of cells.
Therefore, there have been many efforts to overcome these problems, and most of them are directed to modify the native tropism of a virus to infect a specific cell or to express transgenes in only specific cell, using a cell type specific promoter. However, there have been no satisfying results in any of those efforts.